The present invention relates to compounds useful as cytokine production inhibitors, to the preparation of these compounds and to pharmaceutical and veterinary compositions containing them.
It has now been found that fermentation of a strain of the fungus Cladosporium cf. cladosporioides in a nutrient medium produces biologically active novel compounds. Biologically active derivatives of those compounds, which are also novel, can be produced by standard synthetic procedures.
The present invention therefore provides a compound which is a benzofluoranthene derivative of formula (I) 
wherein X is xe2x95x90O, xe2x95x90Nxe2x80x94OR6, NHR or OH wherein R6 is H or C1-C6 alkyl, bond a is oriented  or  and R is H, C1-C6 alkyl which is unsubstituted or substituted by C6-C10 aryl, or C3-C6 cycloalkyl;
 is a bond and  and  are not bonds or, when X is xe2x95x90O,  and  are both bonds and  is not a bond;
R1 and R2, which are the same or different, are H or a halogen;
R3 and R4, which are the same or different, are H, C1-C6 alkyl, C3-C6 cycloalkyl, a heterocyclic group or an aromatic group;
bond e is oriented  or ; and
R5 is C1-C6 alkyl;
or formula (Ib): 
or formula (II) 
or formula (III) 
or a stereoisomer of a said derivative; or a pharmaceutically acceptable salt or ester of a said derivative or stereoisomer.
A C1-C6 alkyl group is straight or branched and is typically C1-C4 alkyl, such as methyl, ethyl, i-propyl, n-propyl, s-butyl, n-butyl or t-butyl. A C3-C6 cycloalkyl group may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. A halogen is F, Cl, Br or I. Preferably it is Cl, Br or I.
An aromatic group is typically C6-C10 aryl. A C6-C10 aryl group may be, for instance, phenyl or naphthyl.
A heterocyclic group is a saturated or unsaturated 5- or 6-membered heterocyclic ring which contains one or more heteroatoms selected from O, N and S and which is optionally fused to a benzene ring or to a second 5- or 6-membered heterocyclic ring. The term heterocyclic group also embraces a heterocyclyl-C1-C6 alkyl group namely a saturated or unsaturated heterocyclic ring as defined above which is linked via a C1-C6 alkylene chain to the point of connection in formula (1).
A saturated heterocyclic ring may be, for example, a tetrahydrofuran, tetrahydropyran, pyrrolidine, piperidine, morpholine or piperazine group. An unsaturated heterocyclic group may be, for example, a furan, thiophene, indole, isoindole, pyrazole, imidazole, isoxazole, oxazole, isothiazole, thiazole, pyridine, quinoline, quinoxaline, isoquinoline, thienopyrazine, pyran, pyrimidine, pyridazine, pyrazine, purine or triazine group. A heterocycyl-C1-C6 alkyl group may be, for example, a furfuryl, pyridylmethyl, pyrrolidinylmethyl or tetrahydropyranylmethyl group.
The heterocyclic group may be unsubstituted or substituted by one or more substituents, for instance one or more substituents selected from OH, halogen C1-C6 alkyl which is unsubstituted or substituted, for example by halogen, such as CF3, C1-C6 alkoxy, nitro and an amino group N(R10R11) as defined above.
In a first aspect formula (I) takes the following definitions; X is xe2x95x90O; R1 and R2 are both H,  is a bond and  and  are not bonds; R3 and R4 are both H; and R5 is C1-C6 alkyl, preferably CH3.
In a second aspect of formula (I) X is NHR as defined above; R1 and R2 are H;  is a bond and  and  are not bonds; R3 and R4 are H; and R5 is C1-C6 alkyl, preferably methyl.
In a third aspect of formula (I) X is xe2x95x90O; R1 and R2, which are the same or different, are H or halogen provided they are both H when R3 is other than H;  is a bond and  and  are not bonds; R3 is H, C1-C6 alkyl, C3-C6 cycloalkyl, a heterocyclic group or an aromatic group; R4 is H and R5 is C1-C6 alkyl, preferably CH3. Preferably R3 is C1-C6 alkyl, more preferably CH3.
In a fourth aspect of formula (I) X is xe2x95x90O;  is a bond and  and  are not bonds; R1, R2 and R3 are H; R4 is C1-C6 alkyl, C3-C6 cycloalkyl, a heterocyclic group or an aromatic group; and R5 is C1-C6 alkyl, preferably CH3, C2H5 OR C3H7. Preferably R4 is C1-C6 alkyl, more preferably CH3.
In a fifth aspect of formula (I) X is xe2x95x90O or OH as defined above;  and  are bonds and  is not a bond; R1, R2, R3 and R4 are H; and R5 is C1-C6 alkyl, preferably CH3.
In formulae (I) and (Ib) bond e at position C8 is preferably on the same side of the molecule as the group at position C7, (OR5 or OCH3 respectively).
In one preferred aspect of the invention the compound is of formula (Ixe2x80x2) 
wherein bond f is oriented  or ; or is a derivative of formula (II) or (III) as defined above.
In a more preferred aspect the compound of formula (I) is of formula (Ia): 
The stereochemical configuration shown in formulae (I), (Ixe2x80x2),(Ia) and (Ib) as depicted above is relative, not absolute. The bonds depicted as being oriented  at C6, C7 and C8 could therefore be  instead whilst those shown as  could be oriented  instead. Diastereoisomers of these structures represent a further aspect of the invention.
The substitution patterns of preferred compounds within formula (I) are shown in the following table:
Some of these compounds are:
3-Cyclopropylamino-7-methoxy-1,2,3,6b, 7,8-hexahydrobenzo[d]fluoranthen-4,8,9-triol (compound 5);
3-Benzylamino-7-methoxy-1,2,3,6b,7,8-hexahydro-benzo[j]fluoranthene-4,8,9-triol (compound 6);
4,8,9-Trihydroxy-7-methoxy-1,6b,7,8-tetrahydro-2H-benzo[j]fluoranthen-3-one oxime (compound 7);
5-Chloro-4,8,9-trihydroxy-7-methoxy-1,6b,7,8-tetrahydro-2H-benzo[j]fluoranthen-3-one (compound 8);
4,9-Dihydro-7-methoxy-8-propoxy-1,6b,7,8-tetrahydro-2H-benzo[j]fluoranthen-3-one (compound 9);
8-Ethoxy-4,9-dihydroxy-7-methoxy-1,6b,7,8-tetrahydro-2H-benzo[j]fluoranthen-3-one (compound 10); and
4,9-Dihydroxy-7,8-dimethoxy-1,6b,7,8-tetrahydro-2H-benzo[j]fluroanthen-3-one (compound 11).
The compounds of formulae (Ia), (Ib), (II) and (III) above have been isolated from a fungus which has been designated Culture Collection Number X20700.
This is a fungus which was isolated directly from a dead insect which had been infected by a member of the entomogenous fungus genus Hypocrella Sacc. The insect was collected from tropical rainforest in Thailand during 1989. A subculture of the isolated fungus was deposited by Xenova Discovery Limited of 545 Ipswich Road, Slough, Berkshire, SL1 4EQ, United Kingdom under the Budapest Treaty at the Centraalbureau voor Schimmelcultures, Baarn, The Netherlands, on Nov. 25, 1997 under reference X15/81/700. It was assigned the accession number CBS 100220.
The fungal strain CBS 100220 is a hyphomycete assigned to the widespread genus Cladosporium Link ex Fr. and no sexually reproducing state was observed in culture. Cultures incubated at 24xc2x0 C. had relatively slowly extending mycelium which attained 6-7 mm diameter after 7d on both 2% MEA and potato/carrot (PCA) agar. No growth was observed on these media at 37xc2x0 C. The recipes for MEA and PCA are given in Smith, D. and Onions, A. H. S., 1983; The Preservation and Maintenance of Living Fungi. Farnham Royal: CABI (using 20 g not 2 g of grated carrot).
Macroscopic characteristics were as follows:
Generally, all cultures sporulated very sparsely within the aerial mycelium. Mycelial margins were submerged on both media whereas the central regions were radially sulcate on MEA only.
Microscopically, the conidiophores were usually septate with thick brown walls at their base tapering and becoming progressively thinner-walled and paler towards their apices. The apices were usually slightly swollen at the point of attachment of the lowermost conidia (ramo-conidia). Ramo-conidia were very rarely 1-septate and formed the main branches of a loose head of branched acropetally extending chains of aseptate conidia with apiculate ends.
Microscopic characters were as follows:
Although the fungus exhibits most of the characters of Cladosporium cladosporicides (Fres.) de Vries, nevertheless it has a slower mycelial extension rate and much sparser conidiogeniesis than usually encountered within this taxon. Furthermore, four other isolates originating from similar source material (two collected during the following year in 1990) had characters consistent with the above details. Hence this fungus has been assigned to Cladosporium cf. cladosporioides. 
The above description is illustrative of a strain of Cladosporium cf. cladosporioides which can be employed in the production of compounds of the present invention. However, the present invention also embraces mutants of the above described microorganism. For example, those which are obtained by natural selection or those produced by mutating agents including ionising radiation such as ultraviolet irradiation, or chemical mutagens such as nitrosoguanidine or the like treatments, are also included within the ambit of this invention.
The present invention further provides a process for producing a compound of formula (Ixe2x80x2), (Ia), (Ib), (II) or (III) as defined above, which process comprises (i) fermenting, in a fermentation medium which provides a source of carbon, nitrogen and inorganic salts, fungal strain X20700 (CBS 100220) or a mutant thereof which produces a said compound; and (ii) isolating the said compound from the fermentation medium.
The invention also provides a biologically pure culture of the fungal strain Cladosporium cf. cladosporioides X20700 (CBS 100220) or a mutant thereof which produces a compound of formula (Ixe2x80x2) (Ia), (Ib), (II) or (III) as defined above. Such cultures are substantially free from other microorganisms. The invention further provides a process for fermenting the fungal strain Cladosporium cf. cladosporioides X20700 (CBS 100200) which process comprises fermenting the said fungal strain or said mutant in a fermentation medium which provides a source of carbon, nitrogen and inorganic salts.
Assimilable sources of carbon, nitrogen and minerals may be provided by either simple or complex nutrients. Sources of carbon will generally include glucose, maltose, starch, glycerol, molasses, dextrin, lactose, sucrose, fructose, carboxylic acids, amino acids, glycerides, alcohols, alkanes and vegetable oils. Sources of carbon will generally comprise from 0.5 to 10% by weight of the fermentation medium.
Sources of nitrogen will generally include soya bean meal, corn steep liquors, distillers"" solubles, yeast extracts, cottonseed meal, peptones, ground nut meal, malt extract, molasses, casein, amino acid mixtures, ammonia (gas or solution), ammonium salts or nitrates. Urea and other amides may also be used. Sources of nitrogen will generally comprise from 0.1 to 10% by weight of the fermentation medium.
Nutrient mineral salts which may be incorporated into the culture medium include the generally used salts capable of yielding sodium, potassium, ammonium, iron, magnesium, zinc, nickel, cobalt, manganese, vanadium, chromium, calcium, copper, molybdenum, boron, phosphate, sulphate, chloride and carbonate ions.
An antifoam may be present to control excessive foaming and is added at intervals as required.
Fermentation can be conducted at temperatures ranging from 20xc2x0 C. to 40xc2x0 C., preferably 24-30xc2x0 C. For optimal results, it is most convenient to conduct these fermentations at a temperature in the range 24-26xc2x0 C. The starting pH of the nutrient medium suitable for producing the compounds can vary from 5.0 to 8.5 with a preferred range of from 5.0 to 7.5.
Small scale fermentations are conveniently carried out by placing suitable quantities of nutrient medium in a flask by known sterile techniques, inoculating the flask with either spores or vegetative cellular growth of the fungal strain, loosely stoppering the flask with cotton wool, and permitting the fermentation to proceed in a constant room temperature of about 25xc2x0 C. on a rotary shaker at from 95 to 300 rpm for 2 to 10 days. The fermentation may also be conducted in static culture on liquid or semi-solid medium.
For larger scale work, it is preferable to conduct the fermentation in suitable tanks provided with an agitator and a means of aerating the fermentation medium. The nutrient medium is made up in the tank after sterilization and is inoculated with a source of vegetative cellular growth of the fungal strain. The fermentation is usually allowed to continue for from 1 to 10 days while agitating and/or aerating the nutrient medium at a temperature in the range 20xc2x0 C. to 37xc2x0 C. The degree of aeration is dependent upon several factors such as the size of the fermenter and agitation speed. Generally the larger scale fermentations are agitated at about 95 to 750 rpm and aerations of about 0.5 to 1.5 VVM (volumes of air per volume of medium per minute).
The separation of the compounds of formulae (Ia), (Ib), (II) and (III) from the whole fermentation broth and their recovery is carried out by solvent extraction followed by application of chromatographic fractionations with various chromatographic techniques and solvent systems. The present compounds in pure form have thus been isolated in this way.
The compound of formula (Ia) can be used as a starting material for the synthesis of structural analogues which are also biologically active. Accordingly, the present invention further provides a process for producing a compound as defined above, which process comprises:
(a) treating a compound of formula (Ia) 
xe2x80x83with an amine of formula Rxe2x80x2xe2x80x94NH2 in which Rxe2x80x2 is H, C1-C6 alkyl which is unsubstituted or substituted by C6-C10 aryl, or C3-C6 cycloalkyl, in water or an organic solvent in the presence of a reducing agent at a pH of from 5 to 6, to obtain a compound of formula (I) in which X is a NHR as defined above; or
(b) treating a compound of formula (Ia) as defined above with a halogenating agent to obtain a compound of formula (I) in which one or both of R1 and R2 is a halogen; or
(c) treating a compound of formula (Ia) as defined above with an alcohol of formula R41xe2x80x94OH in which R41 is as 15 defined above for R4, other than hydrogen, in the presence of an acid, to obtain a compound of formula (I) in which R4 is as defined for R41; or
(d) treating a compound of formula (Ia) as defined above with a halide of formula R31xe2x80x94Y in which Y is a halogen and R31 is as defined above for R3, other than hydrogen, in an organic solvent in the presence of a base and, optionally, a quaternary ammonium halide to obtain a compound of formula (I) in which R3 is as defined above for R31; or
(e) treating a compound of formula (Ia) as defined above with hydroxylamine in an organic solvent and/or water to obtain a compound of formula (I) in which X is xe2x95x90Nxe2x80x94OH; or
(f) treating a compound of formula (Ia) as defined above with a peracid in an organic solvent to give a compound of formula (I) in which  and  are both bonds and  is not a bond; or
(g) treating a compound of formula (Ia) as defined above with a reducing agent in an organic solvent to give a compound of formula (I) in which X is OH as defined above; or
(h) treating a compound of formula (Ia) as defined above with an oxidising agent in to obtain a compound of formula (II) as defined above in which the groups xe2x80x94H and xe2x80x94OCH3 are both oriented , and/or, if desired;
(i) converting a compound obtained in any one of steps (a) to (h) into a pharmaceutically acceptable salt or ester.
Process embodiment (a) is a reductive amination. The reducing agent may be. for example, sodium borohydride or sodium cyanoborohydride. The solvent may be water or an organic solvent which is preferably a polar protic solvent such as an alcohol, for instance methanol or ethanol. The process typically comprises dissolving the compound of formula (Ia) in the organic solvent and then adding the amine Rxe2x80x2xe2x80x94NH to the resulting solution, followed by stirring at about 25xc2x0 C. for 5 to 30 minutes. The reducing agent is then added and the pH of the solution is adjusted to within the desired range by using a drop of bromocresol green in the reaction. The reaction solution may then be concentrated in vacuo and the residue taken up in a suitable organic solvent, for instance dichloromethane or ethyl acetate, and then extracted with water and saturated brine.
In process embodiment (b) the halogenating agent and the reaction conditions are chosen according to whether mono- or di-halogenation is required. To produce a compound of formula (I) in which R1 is a halogen and R2 is hydrogen the halogenating agent is typically a thionyl halide, for instance thionyl chloride or thionyl bromide. The reaction may be conducted at a temperature of about 25xc2x0 C. for a period of from 1 to 8 hours, preferably about 4 hours. To produce a compound of formula (I) in which both R1 and R2 are a halogen, or R2 is a halogen and R1 is hydrogen, the halogenating agent is typically an N-halosuccinimide such as N-chlorosuccimimide, N-bromosuccinimide or N-iodosuccinimide. The reaction is generally conducted in an organic solvent such as a halogenated hydrocarbon, for instance dichloromethane, at a temperature of about 25xc2x0 C. for a period of from 3 to 16 hours.
In process embodiment (c) the compound of formula (Ia) is usually dissolved in the alcohol R41xe2x80x94OH, and the acid is then added dropwise. The acid is generally a concentrated mineral acid such as H2SO4. The reaction mixture is then stirred from 25xc2x0 C. for a period of from 1 to 5 hours, preferably about 2 hours, and may then be neutralised with an alkali such as sodium hydroxide. The desired ether product may then be extracted and purified by conventional methods, for instance by preparative HPLC.
In process emobodiment (d) the halide of formula R31xe2x80x94Y is preferably an iodide and the quaternary ammonium halide is preferably a quaternary ammonium iodide such as tetrabutylammonium iodide. The organic solvent is typically an aprotic solvent such as dimethylformamide, acetonitrile or a halogenated hydrocarbon such as dichloromethane and the base may be, for instance, an alkali metal carbonate such as potassium carbonate or sodium carbonate. The process is generally conducted by dissolving the compound of formula (Ia) in the solvent and then adding thereto the quaternary ammonium salt (trace) followed by the halide. The reaction is then stirred with a saturated solution of the base for a period of 1 to 6 hours, preferably about 2 hours. A further amount of the halide may then be added, if desired or necessary, and the stirring continued. The reaction mixture is then typically acidified, for instance with a strong mineral acid such as HCl or H2SO4, to a pH of about 1. The solvent phase may be separated off and the desired product recovered.
In process embodiment (e) the reaction may be carried out using a salt of hydroxylamine such as hydroxylamine hydrochloride in the presence of sodium hydroxide. The hydroxylamine then reacts as it is released from its salt. The hydroxylamine hydrochloride is typically dissolved in aqueous sodium hydroxide and the pH is adjusted to about 5. The compound of formula (Ia) dissolved in the organic solvent is then added to the solution. The organic solvent is typically an alcohol such as methanol or ethanol. The reaction mixture may be left to stir at a temperature of about 25xc2x0 C. for a period of from 1 hour to 24 hours until the reaction is complete.
Process embodiment (f) involves epoxide formation at the olefinic double bond. The compound of formula (Ia) is typically dissolved in the organic solvent, which may for instance be a halogenated hydrocarbon such as chloroform, and the peracid is added to the resulting solution. Examples of suitable peracids include m-chloroperbenzoic acid and peracetic acid. The reaction mixture is usually stirred at a temperature of about 25xc2x0 C. for a period of from 0.5 to 3 hours to allow the reaction to proceed to completion. The solvent may then be removed and the product purified.
In process embodiment (g) the compound of formula (Ia) is usually dissolved in the organic solvent and the reducing agent then added to the resulting solution. The solvent is preferably an alcohol such as methanol or ethanol. The reducing agent is chosen from those which are suitable for the reduction of ketones to alcohols, such as LiAlH4, LiBH4, NaBH4 and KBH4. NaBH4 is preferred. The reaction mixture is usually left for about 1-2 hours at 25xc2x0 C. before destroying excess reducing agent, for example by adding a few drops of glacial acetic acid. The crude product may then be concentrated In vacuo and purified by conventional means.
Process embodiment (h) is generally carried out by dissolving compound (Ia) in the organic solvent, adding the oxidising agent to the resulting solution and then stirring the reaction mixture at a temperature of about 25xc2x0 C. for a period of from 5 hours to 30 hours, preferably about 24 hours. The solvent is typically a halogenated hydrocarbon such as dichloromethane. The oxidising agent may be any such agent which is suitable for the oxidiation of a secondary alcohol to a ketone, for instance manganese dioxide. The reaction mixture is typically filtered and the product purified following completion of the reaction.
Compounds of formula (I) may be converted into pharmaceutically acceptable salts, and salts may be converted into the free compound, by conventional methods. Suitable salts include salts with pharmaceutically acceptable, inorganic or organic bases. Examples of inorganic bases include ammonia and carbonates, hydroxides and hydrogen carbonates of group I and group II metals such as sodium, potassium, magnesium and calcium. Examples of organic bases include aliphatic and aromatic amines such as methylamine, triethylamine, benzylamine, dibenzylamine or xcex1- or xcex2-phenylethylamine, amino acids such as arginine, and heterocyclic bases such as piperidine, 1-methylpiperidine and morpholine.
Compounds of formula (I) may also be converted into pharmaceutically acceptable esters. Suitable esters include branched or unbranched, saturated or unsaturated C1-C6 alkyl esters, for example methyl, ethyl and vinyl esters.
The compounds of formulae (I), (II) and (III) are inhibitors of the production of cytokines, specifically IL-2. They are also tyrosine kinase inhibitors. These compounds can therefore be used in the treatment of immunoinflammatory conditions. A human or animal, e.g. a mammal, can therefore be treated by a method comprising administration of a therapeutically effective amount of a compound of formula (I), (II) or (III). These compounds can be used in the treatment of immunoinflammatory conditions such as rheumatoid arthritis, osteoarthritis, septic shock, psoriasis, inflammatory bowel disease, Crohn""s disease and asthma, and for the prevention of organ transplant rejection. As tyrosine kinase inhibitors, the compounds of formulae (I), (II) and (III) can be used in the treatment of cancer such as leukemia, malignant melanoma, liver cancer, colon cancer or breast cancer.
The compounds of the present invention can be administered in a variety of dosage forms, for example orally such as in the form of tablets, capsules, sugar- or film-coated tablets, liquid solutions or suspensions or parenterally, for example intramuscularly, intravenously or subcutaneously. The present compounds may therefore be given by injection or infusion.
The dosage depends on a variety of factors including the age, weight and condition of the patient and the route of administration. Typically, however, the dosage adopted for each route of administration for adult humans is 0.001 to 10 mg/kg, most commonly in the range of 0.01 to 5 mg/kg, body weight. Such a dosage may be given, for example, from 1 to 5 times daily orally or by bolus infusion, infusion over several hours and/or repeated administration.
The toxicity of the compounds of the invention is negligible so they can safely be used in therapy.
The compounds of the present invention are formulated for use as a pharmaceutical or veterinary composition also comprising a pharmaceutically or veterinarily acceptable carrier or diluent. The compositions are typically prepared following conventional methods and are administered in a pharmaceutically or veterinarily suitable form.
For example, the solid oral forms may contain, together with the active compound, diluents, such as lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants such as silica, talc, stearic acid, magnesium or calcium stearate and/or polyethylene glycols; binding agents such as starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose, or polyvinyl pyrrolidone; disintegrating agents such as starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dye-stuffs; sweeteners; wetting agents such as lecithin, polysorbates, laurylsulphates. Such preparations may be manufactured in known manner, for example by means of mixing, granulating, tabletting, sugar coating, or film coating processes.
Liquid dispersions for oral administration may be syrups, emulsions and suspensions. The syrups may contain as carrier, for example, saccharose or saccharose with glycerol and/or mannitol and/or sorbitol. In particular a syrup for diabetic patients can contain as carriers only products, for example sorbitol, which do not metabolise to glucose or which only metabolise a very small amount to glucose. The suspensions and the emulsion may contain a carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol.
Suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier such as sterile water, olive oil, ethyl oleate, glycols such as propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride. Solutions for intravenous injection or infusion may contain a carrier, for example, sterile water which is generally Water for Injection. Preferably, however, they may take the form of a sterile, aqueous, isotonic saline solution.
Alternatively, the compounds of the present invention may be encapsulated within liposomes.